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A diverse range of techniques are used in research, analysis, development and clinically to detect cells of interest. Manual or automated techniques are available to count cells in specially designed chambers that permit cell numbers in a sample to be evaluated. Cells may be stained with particular stains in order to differentiate between cell types. Histochemical techniques may be applied to further differentiate between cells in a sample. The ability of cells to respond to particular antigens by proliferating or producing cytokines, bind other cells, engulf other cells or move by chemotaxis may also be diagnostic. Cell surface markers are particularly useful for differentiating between cell types and evaluating the number of particular cells in a sample. Many techniques use antibodies to detect the presence of the marker.
Cells of prokaryotic and eukaryotic organisms, in all their different forms, comprise a cell membrane, which separates the contents of the cell from the extracellular environment. The cell membrane is a selective barrier that determines what goes in and out of the cell and which also undertakes complex signalling activities with other cells and molecules in the environment of the cell. The membrane comprises a phospholipid bilayer and most molecules that cross or interact with the bilayer do so with the assistance of proteins inserted into the bilayer that have both cytoplasmic and extracellular portions.
Frequently, a cell's initial interaction with its surroundings occurs via receptors or cell surface associated molecules expressed on the plasma membrane. Activation of these receptors, whether through binding endogenous ligands (such as cytokines or hormones) or exogenous ligands (such as antigens) triggers a biochemical cascade from the membrane through the cytoplasm to the nucleus. A wide range of receptors or cell surface associated proteinaceous molecules are known to mediate cell:cell interactions and cell:molecule interactions. These include, for example, MHC proteins, cytokine, hormone or neurotransmitter receptors, tethered ligands, G-protein coupled receptors, receptor protein tyrosine kinases, receptor protein tyrosine phosphatases, protein-serine/threonine kinases and receptor guanylyl cyclases.
Most of the proteins that mediate cell-cell recognition or antigen recognition in the immune system contain Ig or Ig-like domains, suggesting that they have a common evolutionary history. These include antibodies, T cell receptors, cluster of differentiation (CD) antigens such as the CD4, CD8, and CD28 proteins, the invariant polypeptide chains associated with B and T cell receptors and Fc receptors on lymphocytes and other white blood cells. About half of the proteins that have been characterized on the surface of white blood cells belong to this superfamily. Cells of the immune system thus express a range of cell surface proteins whose identity can be used to evaluate the number and status of different cells in a sample.
B and T lymphocytes, for example, may be identified using antibodies to the constant regions of the B- and T-cell antigen receptors. T-helper and cytotoxic T-cells cells may be identified on the basis of expression of the co-receptor proteins, CD4 and CD8, respectively.
Flow cytometry is a powerful tool for identifying and enumerating cells. The flow cytometer detects and counts individual cells passing in a stream through a laser beam. By examining large numbers of cells, flow cytometry can give quantitative data on the percentage of cells bearing different molecules, such as surface immunoglobulin, which characterizes B cells, the T-cell receptor-associated molecules known as CD3, and the CD4 and CD8 co-receptor proteins that distinguish the major T-cell subsets. Individual cells within a mixed population are tagged with specific antibodies labelled with fluorescent dyes, or for example, by specific antibodies followed by labelled anti-immunoglobulin antibodies. The suspended mixture of labelled cells is then forced through an aperture, creating a fine stream of liquid containing cells spaced singly at intervals. As each cell passes through a laser beam it scatters the laser light, and any dye molecules bound to the cell will be excited and will fluoresce. Sensitive photomultiplier tubes detect both the scattered light, which gives information on the size and granularity of the cell, and the fluorescence emissions, which give information on the binding of the labelled antibodies and hence on the expression of cell-surface proteins by each cell. If two or more antibodies are used, each coupled to a different fluorescent dye, then the data may be displayed in the form of a two-dimensional scatter diagram or as a contour diagram, where the fluorescence of one dye-labelled antibody is plotted against that of a second, with the result that a population of cells labelling with one antibody can be further subdivided on the basis of its reactivity with the second antibody.
Immunoassays are another particularly useful form of assay that exploit the specificity, strength and diversity of antibody-antigen reactions to analyze samples and detect specific components therein. A wide range of immunoassay techniques are available, such as those described in Wild D. “The Immunoassay Handbook” Nature Publishing Group, 2001.
A wide range of methods for the detection of antibody to specific antigens are also known. For example, the enzyme-linked immunosorbent assay (ELISA) and radio-immunoassay (RIA) are routinely used in laboratories. Arrays and high throughput screening methods are also employed. These methods generally require a high level of skill in laboratory techniques.
A variety of methods have also been developed which require little skill and are rapid to perform, and which are therefore suitable for the detection of antibody to specific antigens, and/or the detection of specific antigens, at the point of care. In particular, lateral flow, dipstick and capillary tube kits have been developed to assay for a number of infections including viral infections.
In subjects with an immunodeficiency disease such as AIDS, the level of CD4 expressing T-cells is an indication of when to commence anti-retroviral drug treatment. The virus infects these T cells and ultimately destroys them. Low CD4+ T-cell levels are also an indication of the risk of clinical progression and susceptibility to opportunistic infection.
In one method of detecting CD4 cells, dynabeads coated with anti-CD4 antibodies are used to bind CD4+ T-lymphocytes. Monocytes, that express CD14 and CD4, are excluded from fresh blood samples sample using beads coated with anti-CD14 antibodies. Thereafter, the isolated CD4 T-lymphocytes are lysed, stained with acridine orange and stained nuclei are enumerated by fluorescence microscopy. A “TRAx CD4” test kit is described in Paxton et al., Clin. Diagn. Lab. Immunol., 2(1):104-114, 1995. This kit is an ELISA based method to measure total CD4 in whole blood samples. The antibodies used did not distinguish between cell bound and soluble CD4 (see Lyamuya et al., J. Imm Methods, 195:103-112, 1996). International Publication No. WO 2006/115866 describe an immunochromatographic device for measuring CD4 antigens. However, again there is no disclosure in this document of a capture reagent capable of distinguishing between cell bound and soluble CD4 lacking a cytoplasmic domain in sample from a subject. Further, the device described in WO 2006/115866 depends upon the flow of sample over a series of numbered capture areas to capture CD4 by saturating consecutive capture areas on a test strip to subsequently provide a visual indication of the concentration of CD4 cells in the sample.
There is a need for improved methods of measuring particular cell types or their cell associated proteins, particularly methods that can be used at point of care and that provide rapid and accurate results.